A Miniaturized, Rapid Technique for Measuring Red Blood Cell-Mediated Microcapillary Occlusion in HbAS, HbSC, and HbSS Sickle Cell Disease Genotypes

Introduction

Sickle cell disease (SCD) is caused by a genetic mutation that produces sickle hemoglobin (HbS). HbS polymerizes upon deoxygenation, causing red blood cells (RBCs) to become sickle-shaped and poorly deformable. Stiff sickle RBCs contribute to microvascular occlusion and organ damage. Previous studies have developed microfluidic devices for assessing RBC-mediated occlusion under hypoxia, but the complexity of the setup and data acquisition limits their adoption. A rapid, easy-to-use, point-of-care assay for assessing RBC-mediated occlusion could increase throughput and aid in determining outcomes of novel therapies, including gene therapies, for SCD. Here, we induced RBC sickling using a chemical method in a simplified workflow to study hypoxic RBC-mediated microcapillary occlusion for healthy (HbAA), sickle cell trait (HbAS), hemoglobin SC disease (HbSC), and homozygous SCD (HbSS) RBCs. Additionally, a custom miniaturized impedance analyzer (MIA) was developed to provide a rapid and fully electronic measurement of occlusion within 10 minutes of sample perfusion.

Methods

Microfluidic devices were fabricated using standard photolithography and polydimethylsiloxane (PDMS) micro-molding protocols. Glass slides were coated with gold electrodes by sputter deposition and bound to the PDMS microfluidic devices using oxygen plasma. The microfluidic devices comprised six microcapillary arrays of widths 12, 10, 8, 6, 4, and 3 μm, with each array coupled with a pair of gold electrodes [3]. The MIA included an ADALM2000 software-defined instrument module, a custom-designed front-end interface board, and a Raspberry Pi single-board computing module. The ADALM2000 was used to generate and acquire a 10-kHz measurement signal, while the front-end interface board handled signal amplification and multiplexed signal routing through the microcapillary arrays. A custom graphical user interface on the Raspberry Pi allowed users to set experimental parameters and monitor data in real time. Venous blood samples were collected in EDTA tubes from participants with HbAS (n=9), HbSC (n=9), HbSS (n=13), and HbAA (n=6) under an IRB-approved protocol. To induce hypoxia, washed RBCs were suspended at 20% hematocrit in a 1.5% (w/v) sodium metabisulphite in 1× PBS buffer and incubated for 3 minutes at room temperature. For normoxia, RBCs from the same donors were suspended at 20% hematocrit in PBS and incubated with the same conditions. Samples were then perfused through the microfluidic device at a constant inlet pressure. After 10 minutes of perfusion, the change in electrical impedance across each array was used to calculate an occlusion index (OI), representing the percent occlusion of the capillary network.

Results

Normoxic OI for HbSS and HbSC was significantly higher than HbAA but there were no statistical differences in HbAA vs. HbAS and HbSC vs. HbSS (Figure 1A). HbAS, HbSC, and HbSS RBCs had significantly higher occlusion under hypoxia than in normoxia (Figure 1A, 1.46 ± 1.67% vs. 8.43 ± 2.78%, P = 0.004 for HbAS, 4.56 ± 2.77% vs. 27.1 ± 17.0%, P = 0.004 for HbSC, and 8.01 ± 6.26% vs. 58.3 ± 26.2%, P = 0.0002 for HbSS). Occlusion of HbAA RBCs remained low in both normoxia and hypoxia (2.70 ± 0.643% vs. 4.72 ± 0.953%, P = 0.0625). Hypoxic OI was significantly different between HbAA vs. HbAS (P = 0.017), HbAS vs. HbSC (P = 0.004), and HbSC vs. HbSS (P = 0.007). Hypoxic OI in HbSC and HbSS was associated with elevated markers of RBC hemolysis - absolute reticulocyte count (ARC, P = 0.0061) and lactate dehydrogenase (LDH, P < 0.0001, Figure 1B).

Conclusion

In normoxia, HbSC had comparable OI to HbSS likely due to HbC induced efflux of potassium ions and water resulting in cellular dehydration and poor RBC deformability. The hypoxic assay, however, unmasked the greater severity of HbSS. Further, elevated hypoxic OI in HbAS may be linked to higher risk of organ damage in this genotype. Chemically-induced hypoxia combined with electrical impedance-based measurement of RBC-mediated microcapillary occlusion offers a new technique for rapidly assessing RBC health and function in a low-oxygen environment. This approach eliminates the need for expensive, high-resolution microscopes and complex gas-exchange chambers. This may serve as a new standard for assessing the clinical efficacy of new treatments that improve RBC deformability and identifying at-risk patients in otherwise mild SCD genotypes.